The N-terminal domain is required for cell surface localisation of VapA, a member of the Vap family of Rhodococcus equi virulence proteins

Rhodococcus equi pneumonia is an important cause of mortality in foals worldwide. Virulent equine isolates harbour an 80-85kb virulence plasmid encoding six virulence-associated proteins (Vaps). VapA, the main virulence factor of this intracellular pathogen, is known to be a cell surface protein that creates an intracellular niche for R. equi growth. In contrast, VapC, VapD and VapE are secreted into the intracellular milieu. Although these Vaps share very high degree of sequence identity in the C-terminal domain, the N-terminal domain (N-domain) of VapA is distinct. It has been proposed that this domain plays a role in VapA surface localization but no direct experimental data provides support to such hypothesis. In this work, we employed R. equi 103S harbouring an unmarked deletion of vapA (R. equi ΔvapA) as the genetic background to express C-terminal Strep-tagged Vap-derivatives integrated in the chromosome. The surface localization of these proteins was assessed by flow cytometry using the THE2122;-NWSHPQFEK Tag FITC-antibody. We show that VapA is the only cell surface Vap encoded in the virulence plasmid. We present compelling evidence for the role of the N-terminal domain of VapA on cell surface localization using fusion proteins in which the N-domain of VapD was exchanged with the N-terminus of VapA. Lastly, using an N-terminally Strep-tagged VapA, we found that the N-terminus of VapA is exposed to the extracellular environment. Given the lack of a lipobox in VapA and the exposure of the N-terminal Strep-tag, it is possible that VapA localization on the cell surface is mediated by interactions between the N-domain and components of the cell surface. We discuss the implications of this work on the light of the recent discovery that soluble recombinant VapA added to the extracellular medium functionally complement the loss of VapA.


Introduction
The actinomycete Rhodococcus equi is multi-host pathogen infecting a wide range of animals as well as immunocompromised humans.It was initially identified as an equine pathogen of young foals causing extensive abscessation and bronchitis of the lung parenchyma [1].However, it has since become clear that R. equi infects a wide range of animals, including pigs and cattle [2].Following uptake by alveolar macrophages, R. equi prevents maturation and acidification of the phagosome in which it resides, eventually causing necrosis of the host cell.Virulent equine isolates of R. equi invariably harbour an 80-85kb virulence plasmid [3], which is necessary replication in both macrophage and mouse models [4,5].The virulence plasmid (pVAPA) contains a 27 kb pathogenicity island (PAI) encoding a highly conserved multigene family of Virulence Associated Proteins (Vap) including vapA, vapC, vapD, vapE, vapG and vapH as well as three vap pseudogenes, vapX, vapI and vapF [6][7][8].In addition to the Vap protein family, the PAI encodes a LysR-type (VirR) and a response regulator (VirS), which are required for expression of the PAI genes and for altering the R. equi transcriptome allowing the pathogen to adapt its physiology to the intracellular niche [6,7,[9][10][11][12]. VapA, together with its transcriptional regulators VirR and VirS, is required and sufficient for intracellular proliferation in macrophages [9,13,14].Despite the high degree of sequence similarity, deletion of vapA can not be complemented by the other vap genes.The presence of VapA results in exclusion of the vATPase and altered proton permeability of the lysosome thus creating a hospitable environment for the pathogen in which it proliferates [15,16].
VapA, C, D, E, H and G have secretion-mediating signal sequences and, with exception of the latter two proteins, have been shown to be secreted into the extracellular environment [6,17].VapA is located on the cell surface of R. equi via an as yet unidentified mechanism [18,19].In contrast, VapC, D and E are present in the supernatant of R. equi cultures, but could not be detected in the cell fraction, suggesting these are not anchored to the cell envelope [17].The Vap proteins contain a highly conserved central and carboxy terminal sequences, which fold into an antiparallel β-barrel formed by eight β-strands separated in the middle by a short α-helix (Fig 1) [20][21][22].Vap proteins are amphipathic due to polar and hydrophobic surfaces along the axis of the β-barrel.In contrast to the highly conserved central and carboxy-terminal sequences, the amino terminal sequences of the mature Vap proteins are highly variable, both in length and composition (Fig 1).Amino-terminally truncated forms of recombinant VapA retain their ability to permeabilise the phagosomal membrane, suggesting that the conserved central and carboxyterminal parts of the protein are required for function [15].Interestingly, the N-termini of VapD, G and B are unordered in crystals suggesting that these are flexible [20][21][22].
To date it remains unclear whether VapA is the only equine Vap protein located on the R. equi cell surface and which residues are important in cell surface anchoring.This study shows that VapA is the only Vap protein located on the cell surface of R. equi.Furthermore, the use hybrid VapA-D proteins suggest a role for the N-terminus of VapA in localisation at the cell surface.

Materials and methods
Bacterial strains, plasmids, oligonucleotides, and growth conditions R. equi 103S containing an unmarked, in-frame deletion of vapA (R. equi ΔvapA) [23] was used as the genetic background for this work.Escherichia coli DH5α (Bethesda Research Laboratories) was used as host for plasmids.Oligonucleotides, plasmids and bacterial strains are listed in Tables 1 and 2. E. coli was grown in lysogeny broth (LB) at 37˚C.R. equi was grown at 200 rpm in LB pH 5.5 at 37˚C (inducing conditions).Agar was added for solid media (1.5%, [w/v]).When appropriate, apramycin was added to media at 80 μg/ml (R. equi) or 50 μg/ml (E.coli) [24].

DNA manipulations
The vap genes were placed under transcriptional control of the vapA promoter (P vapA ), contained within a fragment of 679 bp of the virulence plasmid from the intergenic region upstream of vapA [12].All PCR amplicons generated for downstream cloning were produced with Phusion High-Fidelity Polymerase following manufacturer's recommendations (New

Primer pair Sequence (5' -> 3') a,b
VapA_1249F GCATCTAGAAGACCAACATCGTTCGCG Underlined sequences denote the XbaI restriction site, added for cloning purposes.a Forward primer used together with Vap_ST-rev primer.The number in the right part of the primer name corresponds to the length of the amplification product (bp). https://doi.org/10.1371/journal.pone.0298900.t001 England Biolabs).Amplicons of 1267bp and 1291bp were produced with primer pair VapA_1249F and either VapA_1249R or VapA-ST_1273R (Table 1).The amplicons were cloned in the XbaI site of the integrative vector pSET152 [25] to generate pVapA and pVapA-ST.
For the construction of pVapD and pVapD-ST, primer pair VapA_1249F/VapA_678R was used to amplify a 688bp fragment containing the P vapA and a 503bp fragment containing the vapD gene was amplified with primer pair VapD_495F/VapD_R.The above fragments were joined by blunt-end ligation-dependent PCR with T4 DNA ligase (New England Biolabs) and used as template of amplification with primer pair VapA_1249F/VapD_R or VapA_1249F/VapD-ST_R.The resulting amplicons, of respectively 1191bp and 1215bp, were cloned in the XbaI site of pSET152.Primers VapA_1249F/VapA-SSN_942R were used to amplify a 940 bp DNA fragment, containing P vapA and the vapA sequence encoding both the signal sequence (M1-A31) and the N-terminal region (T32-Q84).Primer pair VapD320_F/VapD_R was used to amplify a 335 bp DNA fragment encoding the C-terminus of VapD (Y57-E164).The above amplicons were joined by blunt-end ligation-dependent PCR using primers VapA_1249F/VapD_R or VapA_1249F/VapD-ST_R.The resulting amplicons of 1275 bp and 1299 bp were cloned in the XbaI site of pSET152 yielding plasmids pVapA::vapD and pVapA::vapD-ST, respectively.Synthetic genes of VapC-ST, VapE-ST, VapG-ST and VapH-ST under P vapA were synthesized by Integrated DNA Technologies and blunt cloned into pUCIDT (Amp) EcoRV site.The design included XbaI sites flanking the synthetic genes for subcloning into the XbaI site of pSET152.All plasmids were purified from E. coli DH5α using the High pure Plasmid isolation and purification kit (Roche) and confirmed by Sanger sequencing using the GATC SupremeRun tubes service (Eurofins, Germany).Electroporation of R. equi 103S ΔvapA, a derivative strain harbouring an in frame deletion of vapA [23] was performed with a GenePulser II coupled to a Pulse Controller Plus (BioRad) as previously described [26].

Flow cytometry
Overnight liquid cultures of R. equi were harvested, washed twice in PBS and their concentration adjusted to OD 600 of 1.0 equivalent to 1.5 x 10 8 cells ml -1 .Aliquots of 5μl containing 7.5 x 10 5 cells were transferred to 1.5 ml tubes and fluorescently labelled with THE 2122; NWSHPQFEK Tag mouse FITC-monoclonal antibody (GenScript, The Netherlands).Briefly, 25 μl of a 5 ng/μl solution of the antibody was added and incubated in the dark for 30 min at room temperature.Unbound antibody was removed by washing as above.After that, 200 μl propidium iodide (PI; BD Biosciences, United Kingdom) was added to a final concentration of 78 ng/μl just before injecting into a CytoFLEX LX flow cytometer (Beckman Coulter Inc).Data was acquired and analysed using the CytExpert 2.4 (Beckman Coulter Inc).The fluorescence of FITC, PI and scattered light of 50,000 cells (or 2 min of recording, whichever occurred first) were simultaneously recorded.Heat-inactivated and unstained controls were used to set up the gates for the differentiation of alive/dead, FITC labelled/non labelled single cells.The population of alive single FITC-stained cells was used for further analysis purposes; unlabelled populations were used for normalization purposes and gating (Fig 2).Proteolytic removal of R. equi surface proteins was performed by 30 min incubation of cells with 0.05% [w/v] trypsin at room temperature.Unless otherwise stated, at least three biological replicates were performed for each strain.Quality control of the instrument was performed daily using the CytoFLEX Daily QC Fluorospheres as per manufacturer specifications (Beckman Coulter Inc).The Stain index (SI) was employed to obtain a measure of the relative brightness of FITC in stained bacterial samples.SI was calculated using the equation: where, F 1 is the median fluorescence intensity of the positive population; F 0 is the median fluorescence intensity of the negative population and SD 0 is standard deviation of the negative population [27].

Infection of J774A.1 cells
Bacteria grown in LB were centrifuged (10 min, 3220 x g) and washed twice with cation-free PBS (Sigma).Murine macrophage-like cells J774A.1 were seeded at 6 x 10 5 cells/ml in 6 cm tissue culture plates (Sarstedt) and cultured at 37˚C in 5% CO 2 overnight.Monolayers were washed once with pre-warmed phagocytosis buffer (0.1% [w/v] gelatin, equal amounts of Medium 199 and DMEM) [28] and the medium was replaced with phagocytosis buffer containing 5% mouse serum (Sigma) as a source of complement.J774A.1 cells were infected with R. equi at a multiplicity of infection (MOI) of 10.Infections were initiated by centrifugation (160 x g, 3 min) of bacteria onto confluent monolayers to synchronize the internalization.
Plates were incubated for 45 min at 37˚C in 5% CO 2 .Monolayers were washed three times with warm phagocytosis buffer (37˚C) to remove unbound bacteria and incubated for a further 15 min to allow internalization of the bacteria attached.Monolayers were washed again with warm phagocytosis buffer.Phagocytosis buffer was subsequently replaced with DMEM supplemented with 10% (vol/vol) fetal calf serum, 4mM L-glutamine, 1% non-essential amino acids and 10 μg/ml gentamycin (time t = 0).Infected monolayers were harvested 0, 24 and 48 h post infection.Medium was replaced after 24 hours with fresh medium containing 10 μg/ml gentamycin.Bacterial growth was determined by dilution plating of macrophage lysates.

Triton X-114 extraction of R. equi cell surface proteins, SDS-PAGE and western blot
R. equi cell surface protein was extracted using a previously reported method [19].Briefly, R. equi cultures grown to stationary phase in LB under VapA inducing conditions were harvested by centrifugation at 3,200 x g for 10 min at 4 ᵒC.Cells were washed twice with sterile TBS buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5).Wet cell pellets were weighted, and 1 ml TBS buffer was added per 50 mg wet cells supplied with 2% (v/v) Triton X-114 and 1 mM PMSF.The resulting cell suspension was extracted overnight at 4ᵒC while rotating the mixture.The cell pellets were removed by centrifugation at 14,400 x g for 10 min at 4ᵒC.The supernatant was incubated at 37ᵒC for 10 min until it became cloudy.Hydrophobic and aqueous phases were separated by centrifugation at 12,000 x g for 10 min at room temperature.The lower Triton X-114 hydrophobic phase was precipitated with 5 volumes of icecold acetone and well mixed and precipitated overnight at -20ᵒC.Precipitated proteins were collected by centrifugation at 4 ᵒC at 12,000 x g for 10 min.The pellets were air dried and subsequently dissolved in 100 μl Tris-HCl (100 mM, pH 8.0), and stored at -20 ᵒC.Protein samples were mixed with 2X loading buffer, denatured for 10 min at 99 ᵒC, loaded into 12% SDS-PAGE gels casted using the BioRad Mini SDS-PAGE Gel system.The BenchMark™ Prestained Protein Ladder (Invitrogen) was routinely used as a molecular weight marker.Immobilon-P polyvinylidene difluoride (PVDF; Millipore) membranes were soaked in methanol for 10 sec and quickly moved to transfer buffer (25 mM Tris, 20 mM glycine, 20% [v/v] methanol).Protein transfer was carried out for 1 hour in an ice bath using transfer buffer at 120 mA using the Mini Trans-Blot Cell System (BioRad).Membranes were blocked with 5% (w/v) fat-free milk (Marvel) in TBST (pH 7.6 10 mM Tris-HCl, 150 mM NaCl, pH 8, 0.1% [v/v] Tween20) for 1 hour at room temperature or overnight at 4 ᵒC.After three washing steps with TBST, membranes were incubated with both the primary mouse anti-VapA monoclonal antibodies [29] and secondary anti-mouse IgG (H+L), HRP Conjugate (Promega), at dilution of 1:5000 and 1:1000 respectively.Membranes were washed a final time with TBST and developed using the Lumi-Light Western Blotting Substrate Kit according to the manufacturer's recommendations (Roche).Western blots were visualised using the Fluorchem FC2 Imaging System (Alpha Innotech).

Data analysis
Statistical analysis was performed in GraphPad Prism10 using the Brown-Forsythe and Welch ANOVA test and corrected P values for multiple comparisons with the Dunnet's T3 test.

Addition of a C-terminal Strep-tag does not affect VapA functionality
To facilitate analysis of the localisation of Vap proteins we introduced a Strep-tag at the C-terminal end of the vap genes which were expressed from the P vapA promoter on the integrative plasmid pSET152.To demonstrate that the C-terminal Strep-tag did not affect the association with the cell envelope, R. equi ΔvapA/pVapA-ST and the wild-type strain were grown in LB under vapA inducing growth conditions and subjected to Triton X-114 extraction, followed by western blot analysis using anti-VapA monoclonal antibodies and anti-Strep antibodies.VapA and VapA-Strep could both be detected in the Triton X-114 phase using anti-VapA antibodies (  The PAI of pVAP1037 encodes five additional Vap proteins, VapC, D, E, G, H.To examine whether these are also associated with the cell surface, C-terminal Strep-tag fusions were created.The resulting constructs which retained their 5' regions including the ribosome binding sites were cloned downstream from the P vapA promoter on pSET152 and inserted into the chromosome of R. equi ΔvapA.To confirm that the vap-ST genes were expressed, RNA was isolated following growth under conditions that induce the P vapA promoter and used as template in RT-PCR using primers specific for vapC,D,E,H and G (Fig 6).This demonstrated that all vap constructs were transcribed.
In contrast to cells expressing VapA-ST that were labelled with FITC-antibody (Fig

Fusion proteins of VapD containing the N-domain of VapA are expressed on the cell surface
In contrast to VapA, VapD, which has the shortest N-terminal sequence, is not located on the cell surface (Fig 7C ) and is secreted into the extracellular environment [17].In contrast to the highly conserved nature of the middle and C-terminal parts of the Vap proteins, the N-terminal sequences of Vap proteins are highly variable.Given that the VapA N-terminal sequence is the longest of the Vap proteins and that VapA is the only Vap associated with the cell surface, we hypothesized that the VapA N-terminal sequence is involved in directing VapA to the cell In both cases, the stain index of trypsinised cells was significantly lower than the undigested cells (0.386 ± 0.142, P = 0.0478 and 0.137 ± 0.141 P = <0.0001,respectively).Taken together, our findings produced compelling evidence that the N-terminus domain of VapA plays a role in the localization of VapA on the cell surface.

The VapA-VapD hybrid protein does not support R. equi intracellular growth
Replacement of the VapD terminal domain with that of VapA resulted in cell surface localisation of VapD and offered the possibility that the hybrid VapA-VapD protein could be

The N-terminal domain of VapA is exposed to the extracellular environment
The results show that the N-terminal domain of VapA plays a role in the cell surface localisation.A possible mechanism is the insertion of the N-terminal domain into the cell envelope, thus providing an anchor for VapA.To investigate the accessibility of the N-terminus of VapA to the extracellular environment, we generated a derivative of VapA containing the Strep-tag inserted between residues A 31 and T 32 , which corresponds to the cleavage site of the signal peptidase as predicted by SignalP and corroborated by Edman sequencing [19].In this configuration, it is the Strep-tag that becomes the N-terminus of the mature VapA protein.Flow cytometry analysis of cells of R. equi 103S ΔvapA/pST-VapA grown under vapA inducing conditions and stained with the anti-Strep tag FITC-antibody resulted in a population of fluorescent cells with SI of 4.92 ± 1.13, which decreased to 1.61 in cells that were pre-digested with trypsin (Fig 9).These results demonstrate that the N-terminus of VapA is accessible to the extracelluar environment.In addition, these results show that the presence of a N-terminal threonine is not a requirement for cell surface localization.

Discussion
It has been known for quite some time that VapA is a cell surface associated protein.Trypsination of intact R. equi cells rendered VapA undetectable in western blots.Other methodological approaches that have been used to confirm the localization of VapA on the cell surface are aqueous two-phase extractions of whole cells with Triton X-114, in which VapA partitions into the hydrophobic phase as judged by detection with anti-VapA antibodies [19] and flow cytometry of R. equi cells using either polyclonal or monoclonal anti-VapA antibodies, followed by detection using a FITC-conjugated secondary antibody [5,18,30].This study confirms these earlier studies showing that VapA is indeed surface localised.
The mechanism by which VapA is associated with the surface of the cell-envelope remains unknown.It has been suggested that VapA is a lipoprotein based on radio-labeling experiments using [9, 10-3 H] palmitate [19].However, VapA does not contain a lipobox: a conserved amino acid sequence at the carboxy terminal end of the signal sequence, followed by a conserved cysteine residue.The invariant cysteine residue of the lipobox forms a thioether bond with lipid by preprolipoprotein diacylglyceryltransferase. The lipid modified preproprotein serves as substrate for signal peptidase II, which removes the signal sequence and leaves the modified cysteine residue at the amino terminus of the mature protein [31][32][33].It has since been shown that R. equi is able to metabolise palmitate [10], which suggests that radiolabeling of VapA is not due to lipidation by palmitate but by incorporation of 13 C-labeled amino acids derived from palmitate metabolism.VapA is highly expressed, and, as a result, a substantial percentage of the 13 C-label would be incorporated into this protein.
A novel lipobox-independent, N-terminal glycine acylation system was recently discovered in E. coli, widening the repertoire of protein acylation mechanisms involved in protein anchorage of cell surface proteins [34].However, the mature VapA protein contains an N-terminal Thr rather than an Gly residue.To date there is no known mechanism for lipidation of N-terminal Thr residues.Furthermore, introduction of an N-terminal Strep-tag which replaced the N-terminal Thr residue with Trp did not affect surface localisation of VapA, demonstrating that the N-terminal Thr residue is not essential for localisation.We therefore conclude that unless R. equi possesses an as yet unidentified protein lipidation mechanism, it is unlikely that VapA is a lipoprotein.Furthermore, VapA does not contain a C-terminal LPXTG motif which in Gram-positive bacteria is used to covalently link proteins with the peptidoglycan layer [35].
Several studies have shown that VapA interacts with host membranes [15,36] resulting in permeabilisation of the phagosomal and lysosomal membranes thus preventing acidification of these compartments [15].Interaction of VapA with host membranes does not depend on the presence of its amino terminal portion, since core-VapA protein lacking this sequence not only retains its ability to bind to host membranes, but also retains functionality [15].Structural analysis of VapD, VapB and VapG showed that the core-Vap structure is amphiphatic, containing a 'bald' spot that is devoid of side chains due to the presence of several glycine residues and is surrounded by an extensive non-polar region [20][21][22].
The VapD structure showed the presence of two octyl-β-d-glucoside molecules using during crystalization bound to this apolar region, suggesting it may playa role in directing Vap proteins to ordered lipid structures in host membranes or to glycolipids, e.g., trehalose 6,6 0dimycolate, present in the cell envelope of R. equi [20].A recent structural study of VapB revealed the presence of a potential ligand-binding site in the apolar region of Vap proteins that may faciliate interaction with lipids [37].However, our study shows that in contrast to VapA, VapD does not bind to the R. equi cell envelope, showing that although the apolar region of Vap proteins may be important in interaction with the cell envelope, it is not sufficient.In silico analysis of the N-terminal Vap sequences suggested, and circular-dichroism spectroscopic analysis of VapD showed, that the N-terminus of Vap proteins is disordered and flexible [20].Given that the main difference between Vap proteins is their highly divergent Nterminal sequence, we hypothesised that the N-terminus of VapA is critical for the unique function or localisation of VapA.Since the core-VapA protein retains it ability to permeabilise phagosmal and lysosomal membranes [15], we explored whether it plays a role in surface anchoring of VapA.The data presented here show that the disordered N-terminal domain of the mature VapA protein confers to VapD the ability to bind to the surface of R. equi.However, the VapA-VapD fusion protein could not restore intracellular growth of a vapA deletion mutant, suggesting that although surface localisation and enabling intracelluar growth are two unique features of VapA they are not dependent on each other.
The VapA protein together with the transcriptional regulators VirR and VirS is essential and sufficient for growth of R. equi 103S in murine macrophages [9].The PAI of the virulence plasmid encodes a further five Vap proteins, which despite their high degree of sequence similarity with VapA do not complement a vapA deletion mutant and restore intramacrophage growth [9,13].In addition to its unique cell surface location, VapA also differs from the other Vap proteins by its high expression level [12,38].The vapA gene is cotranscribed into a four cistronic vapAICD mRNA, which is processed into a stable vapA and an unstable vapICD transcript, which is a likely explanation for its distinct high level of expression [24,39].R. equi vapA deletion mutants are not capable of intracellular growth, however, they can be rescued by the addition of purified soluble VapA [14,36].Subsequent reports showed that soluble VapA reaches lysosomes early during R. equi infection, and that soluble VapA increases the pH of the lysosome by inducing small lesions in the membrane [15].However, high, nonphysiological, concentrations of soluble VapA (10-100 μg/ml) were required to rescue intracellular growth of the vapA mutants.A possible explanation for the surface localisation of VapA is that it may provide R. equi with a mechanism to achieve high local concentrations of VapA required for productive interactions with the host membrane.
Many bacteria, including R. equi and M. tuberculosis, produce extra-cellular vesicles (EVs) of approximately 20-500 nm in diameter, that play an important role in pathogenesis and host-pathogen interaction [40][41][42].R. equi encodes homologues of the dynamin-like proteins IniA (REQ_40370; 50% identity, 67% similarity) and IniC (REQ_40360; 55% identity, 67% similarity), which are required for EV synthesis in M. tuberculosis [43].Furthermore, R. equi encodes homologues (REQ_37790 and REQ_37800) of the Pst/SenX3-RegX3 two-component regulatory system as well as a homologue of VirR (REQ_38840) which regulate EV biosynthesis in M. tuberculosis [44,45].The mechanism for EV formation in R. equi and M. tuberculosis may therefore be similar.EVs may contain cytoplasmatic content as well as components of the cell envelope.These include toxins, siderophores and immune invasion proteins [41,46].Proteomic analysis M. tuberculosis EVs revealed the presence of 287 cell surface, secreted and some cytoplasmic proteins [47].R. equi EVs contain trypsin-susceptible VapA, strongly suggesting that VapA is located on the surface of these vesicles [42].Based on this study we propose the hypothesis that cell surface localisation of VapA is at least in part mediated by its disordered N-terminal domain and serves to facilitate high local VapA concentrations and, in addition, faciliates incorporation in EVs that may play a role in R. equi pathogenesis.

Fig 2 .
Fig 2. Gating strategy.Representative figure of the gating strategy.A gate on time was used to guaranty that the analysis is performed only when the measurement was stable (A).Bacteria were identified and selected based on their scatter properties (using the height measurement of these signals in logarithmic scales) (B).A double gating was used to exclude aggregated events (FSC-H vs FSC-Width and FSC-H vs FSC-A) (C, D).Propidium iodide was used to identify and exclude bacteria with membrane instability (dead) (E).Distribution of the FITC intensity of 50,000 single alive bacteria.https://doi.org/10.1371/journal.pone.0298900.g002

Fig 3 )
. To further confirm that the Strep-tag did not interfere with functionality of the VapA protein, R. equi ΔvapA was complemented with pVapA-ST and used to infect macrophage monolayers.As expected, deletion of the vapA gene prevented intracellular growth of R. equi showing that vapA is essential for intracellular growth.Complementation with pVapA-ST restored intracellular growth, demonstrating that the addition of a C-terminal Strep-tag does not affect functionality of VapA (Fig4).VapA is the only virulence associated protein expressed on the cell surface of R. equi 103SPrevious studies have assessed the cell surface expression of VapA by flow cytometry using anti-VapA antibodies[5,30].Given the lack of antibodies against different members of the Vap family of proteins, we used a commercial anti-Strep-Tag [FITC]-antibody (FITC-antibody) to detect Strep-tagged VapA (VapA-ST) expressed in the background of R. equi 103S ΔvapA.Cells with high levels of fluorescence were observed with R. equi 103S ΔvapA/pVa-pA-ST stained with the FITC-antibody (Fig5A).The fluorescent signal attributed to non-specific binding and to autofluorescence were measured using live R. equi 103S ΔvapA/pVapA incubated with the FITC-antibody (Fig 5B) and R. equi 103S ΔvapA/pVapA-ST incubated in

Fig 3 .
Fig 3. Addition of a C-terminal Strep-tag does not affect cell surface localisation of VapA.R. equi ΔvapA/ pVapA-ST was grown under vapA inducing conditions, followed by extraction with 2% (v/v) Triton X-114.VapA monoclonal antibodies and Strep-tag HRP conjugate antibodies were used to detect VapA.Lane 1: western blot developed using VapA monoclonal antibodies.Lane 2: western blot developed using Strep-tag HRP conjugate antibodies.Bars on the left indicate the molecular mass in kDa.https://doi.org/10.1371/journal.pone.0298900.g003 7A), incubation of R. equi 103S ΔvapA harbouring either pVapC-ST, pVapD-ST, pVapE-ST, pVapG-ST or pVapH-ST (Fig 7B-7F) resulted in SI values in the range of 0.05 to 0.41.These results are consistent with VapA being the only Vap protein encoded on pVAP1037 that is associated with the cell surface.

Fig 4 .Fig 5 .
Fig 4. Complementation of R. equi ΔvapA with pVapA-ST and pVapA::VapD-ST.Murine J774A.1 cells were infected with either R. equi ΔvapA or R. equi ΔvapA carrying either pVapA-ST or pVapA::VapD-ST.Intracellular R. equi were enumerated 0,24 and 48 hours post infection.Intracellular growth is shown as fold changes relative to time zero.Error bars represent the mean and standard deviation.Horizontal lines show the Dunnet's T3 adjusted P value corrected for multiple comparisons.For simplicity only relevant comparisons are shown.https://doi.org/10.1371/journal.pone.0298900.g004

Fig 8 .
Fig 8.The N-terminus of VapA plays a role in surface localization.Graphical representation of the fusion proteins containing the N-terminus of VapA (T 32 -Q 84 ) and the C-terminal domain of VapD that were employed to assess the role of the N-terminus of VapA on surface localization (A).Fusion proteins VapA(SS-N)::VapD-ST (B) and VapD (SS)::VapA(N)::VapD-ST (C).Cell surface proteins were detected with THE 2122; NWSHPQFEK Tag mouse FITCmonoclonal antibody (Red) but not detected when cells were digested with trypsin before incubation with the antibody (Black).https://doi.org/10.1371/journal.pone.0298900.g008

Fig 9 .
Fig 9.The N-terminus of VapA is exposed to the extracellular environment.Detection of the ST-VapA protein on the cell surface by flow cytometry was used to detect accessibility of the N-terminal domain to the extracellular environment (Red).Trypsin digestion of the cells before incubation with the anti-Strep-tag FITC-antibody removed the antibody binding sites (Black).https://doi.org/10.1371/journal.pone.0298900.g009